PB1958: IN SILICO ANALYSIS OF SYNONYMOUS AND NON-SYNONYMOUS SNPS OF THE HUMAN CEREBLON (CRBN) GENE: AN IMPORTANT GENE IN IMMUNOMODULATORY DRUGS (IMIDS)-BASED TREATMENT OF MULTIPLE MYELOMA PATIENTS
Authors: M. Güler, A. Macauda, F. Canzian
Journal: HemaSphere (2022)

Background: Immunomodulatory drugs that target cereblon, a component of the E3 protein ligase complex encoded by the CRBN gene, are used to treat multiple myeloma (MM) and myelodysplastic syndrome. Because the CRBN gene is so important in IMiD metabolism, it is of interest to assess the effects of single nucleotide polymorphisms (SNPs) on cereblon, which can potentially affect IMiD treatment response in MM patients Aims: This study aims to detect deleterious SNPs (i.e. disrupting cereblon function) in CRBN by using bioinformatics tools based on protein-IMiD structure, nucleotide and protein sequence, and post-translational modifications (PTMs). Methods: The CRBN SNPs were collected from dbSNP with filtering validation status by ALFA option. The nucleotide sequence of CRBN and amino acid sequence were downloaded from NCBI and UniProt respectively. The structure of the CRBN-pomalidomide complex was downloaded from RCSB PDB and the CRBN structure was obtained from AlphaFold. Genetic variants were analyzed with different tools for variant prioritization. The missense variants were scored by 12 different tools (PANTHER, SIFT, PolyPhen, CADD, BayesDel, ClinPred, LRT, M-CAP, MetaLR, MetaSVM, REVEL, fathmm-XF) and PTM position. The potential structural effects of amino acid changes were predicted by PremPLI (protein-IMiD complex) and INPS-MD (only protein) tools by prediction of protein stability free energy change (ΔΔG). The non-coding and non-exonic variants were scored by 10 tools (CADD, Deepsea, eigen_PC, fathmm, funseq2, fitcons_Fitness, gerp_Base.RS, gwava, phast, ReMM) and CpG region, TFBS and miRNA binding site. After calculating scores by different tools, we assigned to each SNP deleterious-benign status (0 and 1) for each prediction, then we calculated a Z-score and cumulative distribution function of normal distribution of Z-scores (one-tailed, 95% CI). Results: dbSNP has 12611 CRBN SNPs validated by ALFA: 267 deletions, 1394 deletion-insertions, 78 insertions, and 10872 single nucleotide variants. After removing duplicates, we found 314 non-synonymous and 8602 synonymous and non-coding SNPs. The experimentally detected PTM sites are extremely conserved, as only eight of them have SNPs, all of them being rare in all populations (minor allele frequency between 0-0.00002639). The 27 of 314 non-synonymous SNPs passed nominal p-value (<0.05), but after Bonferroni correction (0.05/314) none of them were significant. However, PremPLI and INPS-MD tools-based prediction of protein stability free energy change (ΔΔG) values were significantly different between these 27 SNP and the others. The INPS-MD predicted ΔΔG mean in significant SNPs is -1.048kcal/mol (min -3.659-max 0.645), while in the non-significant group the mean was -0.376kcal/mol (min -3.361-max 1.617), t-Test p=1.61x10-6. The PremPLI-predicted ΔΔG mean in the top 27 SNPs was 0.415kcal/mol (min 0.01-max 0.76), while in non-significant group the mean was 0.309kcal/mol (min -0.93 - max 0.9), t-Test p=0.041. The 107 of 8602 synonymous and other non-coding SNPs passed p-value threshold after multiple test correction (0.05/8602). The gnomAD global allele frequency of these SNPs is ranging from 1.16x10-4 to 8.34x10-6. Summary/Conclusion: As a result, the PTM sites of cereblon are clearly functionally significant. Therefore, malfunctions in proteins involved in the post-translational modification of cereblon should have an impact on its activity. Furthermore, protein stability ΔΔG predicts also deleteriousness of SNPs. Even though significant SNPs are very rare, they should be tested to assess their functional effect in MM cells.